The long-term goal of this research is to improve the rational design of artificial zinc finger protein (ZFP) transcription factor therapeutics by characterizing the fundamental thermodynamics of ZFP transcription factor interactions with zinc metal-ions and DNA. ZFPs, the largest single class of metalloproteins in the human genome, regulate gene transcription and, therefore, protein expression, by binding both zinc metal-ions and DNA or RNA. Since ZFP transcription factors can be potentially designed to activate or repress any gene in the genome, these proteins are excellent targets for drug design. Indeed the rational design and combinatorial selection of artificial ZFPs is beginning to yield potential drug candidates, one of which is in clinical trials for diabetic neuropathy and Lou Gehrig's disease. Furthermore, recent success in ZFP protein transduction in mammalian cells indicates they may be suitable for gene therapy applications. One issue that slows the progress of artificial ZFP design is coupled energetics of zinc metal-ion binding, DNA-binding, and protein folding in ZFPs. Based on our success in parsing apart the energetics of zinc metal-ion binding and protein folding using simple designed peptides, we are in a unique position to decouple the binding of zinc metal-ions and DNA in a natural ZFP, the Wilms tumor suppressor. We will focus our research efforts in delineating the fundamental thermodynamics of ZFP binding to zinc metal-ions and DNA. Our approach is to measure the equilibrium thermodynamics of Zn(II) and DNA binding to ZFPs. Based on the current lack of understanding of the coupled binding of Zn(II) and DNA to ZFPs, we have identified two Specific Aims to be completed during the requested funding period. Specific Aim 1: Study of zinc finger protein Zn(II) affinity in the presence of DNA. Specific Aim 2: Study of apo- zinc finger protein folding. PUBLIC HEALTH RELEVANCE: The detailed understanding of the interrelationship of Zn(II) and DNA binding to zinc finger proteins provided by this study will lead to a greater understanding of general metalloregulatory processes, and specifically gene activation in human cancer. These results will facilitate both the rational design of tumor suppressor drugs and improved prediction of the process of cancer gene activation. Therefore, benefits to healthcare and the general welfare of the public are anticipated from the successful completion of this work.